Phenylisopropyl Adenosine Research Articles

Cultured vascular smooth muscle cells from porcine coronary artery possess A1 and A2 adenosine receptor activity

This study tested the hypothesis that an A1 adenosine receptor capable of inhibiting adenylate cyclase activity is present in porcine coronary vascular smooth muscle cells. In the absence of blockade of the A2 adenosine receptor, the A1 adenosine receptor agonists phenylisopropyladenosine (PIA) and cyclopentyladenosine (CPA) (10 −9M) failed to inhibit Gpp(NH)p stimulated adenylate cyclase activity. However, after blockade of the A2 adenosine receptor with 30 nM CGS 15943A, cyclopentyladenosine (10 −9M) inhibited Gpp(NH)p stimulated adenylate cyclase activity by 27±3% (4.3±0.7, Mean±SEM; pmoles/min/mg vs 5.9±0.8, P<.05). The data demonstrate that both A1 and A2 adenosine receptors are present in coronary vascular smooth muscle. The results indicate that adenosine may mediate both vasodilation and vasoconstriction in the coronary circulation via A2 and A1 adenosine receptors, respectively.

Reperfusion Injury after 24 Hours Cold Storage of Donor Hearts. Possible Role of Leukocyte Activation

The role of neutrophils on the development of reperfusion injury was evaluated in canine hearts. Reperfusion was performed with a closed reperfusion system. 18 dog hearts were arrested by a single dose of National Institute of Health cardioplegia and stored cold (0.5 degrees C) for 24 hours. Thereafter they were randomly attached to one of the following groups. Group I: reperfusion with whole blood (n = 6), group II: reperfusion with low-leukocyte blood (n = 6), group III: reperfusion with low-leukocyte blood plus phenyl-isopropyl adenosine (PIA) (n = 6). Hearts in group I showed an increase of high energy phosphates (HEP) within the first 30 minutes, and a significant decrease during the subsequent 30 minutes. Concomitantly the initially soft hearts turned into stone hearts. In group II and III a significant increase of HEP was seen during the entire reperfusion. After 60 minutes of reperfusion myocardial ATP levels in group III were significantly higher than in group I. All hearts in group III were soft after reperfusion. It is concluded that reperfusion injury appears to be dependent upon the presence and activation of neutrophils, which can be partially prevented by reperfusion with low-leukocyte blood and more so by the simultaneous administration of PIA.

Respiratory Response to Adenosine in Newborn Lambs Is Modified by Hypoxemia and by Heat Stress

A study was made to determine whether adenosine is involved in the regulation of breathing and whether the respiratory response to injections of adenosine and its analogue L-PIA (phenylisopropyl adenosine) is modified by moderate hypoxemia or by heat stress. Unanesthetized lambs with chronically implanted catheters were used for the investigation. Intravenous injections of adenosine caused an increase in the rate of breathing lasting for 5-30 s, except during heat stress, when the rate of breathing decreased. Injection of L-PIA also increased the rate of breathing, but the effects lasted longer than after injection of adenosine. During heat stress the response to injection of L-PIA varied. Both the arterial blood pressure and heart rate decreased transiently after intravenous injection of adenosine or L-PIA, while the central venous pressure increased. Adenosine might thus be part of a positive feedback system that stimulates breathing under normal environmental conditions with or without moderate hypoxemia. The respiratory responses to injection of adenosine during heat stress are different, which might indicate that under this condition the effects of adenosine on breathing are modified by its influence on temperature control.

Neonatal Caffeine Exposure Alters Adenosine Receptor Control of Locomotor Activity in the Developing Rat

An animal model was developed to approximate the magnitude and brain developmental period of caffeine exposure experienced by the human infant receiving caffeine for apnea of prematurity. Serum levels of caffeine of 5-15 mg/l over the 24-hour period were achieved by administering to rat pups a loading dose of 20 mg/kg p.o. on day 2, followed by 15 mg/kg p.o. on days 3-6 of life. Locomotor activity, both spontaneous and following an acute challenge with D-phenylisopropyl adenosine (an adenosine receptor agonist, 10 mumol/kg i.p.) or caffeine (an adenosine receptor antagonist, 100 mumol/kg i.p.), was measured in 12-to 28-day-old native rat pups or pups neonatally exposed to caffeine. The stimulatory effect of acute caffeine on locomotor activity was developmentally delayed in neonatally exposed pups. It is postulated that early exposure to caffeine in some way altered the development of the central adenosine receptor control over locomotor activity.

The use of selective inhibitors and computer modelling to evaluate the role of specific high affinity cyclic AMP phosphodiesterases in the hormonal regulation of hepatocyte intracellular cyclic AMP concentrations

Using experimentally derived data for the activities and kinetic constants of hepatocyte cyclic AMP phosphodiesterase isoenzymes together with the derived changes in adenylate cyclase activity, due to stimulation and subsequent desensitization by glucagon, a computer model was established to simulate hepatocyte cyclic AMP metabolism. The established ability of glucagon to activate the ‘dense-vesicle’ cyclic AMP phosphodiesterase by eliciting its cyclic AMP-dependent phosphorylation was shown on the model to be capable of eliciting a profound reduction in the glucagon-stimulated increase in intracellular cyclic AMP. This was consistent with experimentally derived observations using the compound ICI 118233 which was used to inactivate the ‘dense-vesicle’ enzyme selectively. The non-hydrolysable adenosine agonist N 6 (phenylisopropyl)-adenosine (PIA), which prevents glucagon pre-treatment of hepatocytes blocking the ability of insulin to stimulate the peripheral plasma membrane cyclic AMP phosphodiesterase, is shown here to accentuate the ability of insulin to decrease glucagon-elevated intracellular cyclic AMP concentrations. This effect was obliterated using the compound ICI 63197, a selective inhibitor of the peripheral plasma membrane phosphodiesteras. Computer modelling studies, taking into account experimentally derived actions of insulin in activating the peripheral plasma membrane phosphodiesterase, confirmed the potential of this enzyme to decrease intracellular cyclic AMP concentrations. Modelling of the putative effect of an insulin ‘mediator’ in activating the two cyclic GMP-stimulated cyclic AMP phosphodiesterase isoenzymes was shown to elicit a decrease in intracellular cyclic AMP concentrations which was comparable to that caused by insulin's action on intact hepatocytes. The relative contribution of each phosphodiesterase form to the metabolism of hepatocyte intracellular cyclic AMP, together with an assessment of the potential effect of inhibition and activation of specific species, was evaluated using the computer model. These experimental and simulation studies indicate that alterations in the phosphodiesterase activity of the ‘dense-vesicle’ enzyme, the peripheral plasma membrane enzyme, the cyclic GMP-stimulated cyclic AMP isoforms and the IBMX-insenstive PDE-MQ-II can elicit profound effects upon hepatocyte intracellular cyclic AMP concentrations.

A selective binding site for 3H-NECA that is not an adenosine A 2 receptor

In homogenates of NG108-15 cells, adenosine analogues activate adenylate cyclase with the following order of potency: N- ethylcarboxamidoadenosine ( NECA) >2- chloroadenosine >N 6-( L- phenylisopropyl)- adenosine (PIA) = cyclohexyladenosine = 2-phenylaminoadenosine. Adenosine receptor antagonists inhibit NECA-stimulated adenylate cyclase activity with the order of potency 3-isobutyl-1-methyl-xanthine (IBMX)>theophyllin > caffeine. These data suggest that these ligands act at an adenosine A 2 receptor. There is an apparently homogenous population of saturable 3H-NECA binding sites in homogenates of NG108-15 cells. These sites have an affinity for 3H-NECA of t~1 ωM, and are present at a density of ≈10 pmol/mg protein. Unlabelled NECA, 2-chloroadenosine, IBMX and theophylline displace 3H-NECA binding, with an order of potency that suggests that the 3H-NECA binding site may represent an adenosine A 2 receptor. However, PIA, cyclohexyladenosine and 2-phenylaminoadenosine produce no detectable displacement of 3H-NECA binding at concentrations that produce a maximal stimulation of adenylate cyclase activity. Pretreatment of NG108-15 cells with either NECA or PIA produces a homologous desensitization of subsequent responses to all the adenosine analogues, with no effect on subsequent responses to a prostacyclin receptor agonist or NaF. This suggests that all the adenosine analogues examined activate an adenosine A 2 receptor. Therefore, the 3H-NECA site at which PIA is inactive cannot represent this receptor.

Quinolinic acid neurotoxicity: protection by intracerebral phenylisopropyladenosine (PIA) and potentiation by hypotension.

In the present study we have used the purine agonist R-phenylisopropyladenosine (PIA) which suppresses excitatory amino acid release to assess its effect on quinolinate toxicity. Quinolinic acid was injected into the rat hippocampus alone or with PIA and the animals allowed to recover. After 4 days the brain was removed for histological examination. The extent of neuronal degeneration was assessed blind by an independent observer on a scale of 0 (no damage) to 10 (complete degeneration). Co-administration of PIA protected against the toxicity, and this protective action of PIA was blocked by a xanthine adenosine receptor antagonist. However, systemic injections of PIA or the non-purine ganglion blocking drug trimetaphan, both of which caused significant depression of blood pressure, potentiated quinolinate toxicity. The results may indicate an interaction between endogenous excitotoxins and episodes of hypotension which may be critical in determining cell death in the CNS.

Interactions between adenylate cyclase inhibitors and beta-adrenoceptors in isolated human fat cells

Interactions between adenylate cyclase inhibitors and beta-adrenoceptors were investigated in isolated human fat cells. Phenylisopropyl adenosine, nicotinic acid and prostaglandine E 2 induced a dose-dependent decrease in beta-adrenoceptor sensitivity; the concentration of isoprenaline causing half-maximum lipolytic effect increased 100-fold. The affinity constants for the high and low affinity beta-adrenoceptor states were increased 3000 and 700 times, respectively, but the total number of binding sites was unchanged. Pertussis toxin caused a dose-dependent increase of beta-adrenoceptor sensitivity to isoprenaline. There was a 200-fold increase in isoprenaline sensitivity in the lipolysis experiments and corresponding increases in the receptor affinity in the binding experiments. It is concluded that the affinity of human fat cell beta-adrenoceptors is reduced by adenylate cyclase inhibitors. This seems to be mediated by the G i-protein and represents a new potential mechanism by which lipolysis is regulated by inhibitors of adenylate cyclase in man.

Stimulation of adenosine receptor enhances α1 -adrenergic receptor-mediated activation of phospholipase C and Ca2+ mobilization in a pertussis toxin-sensitive manner in FRTL-5 thyroid cells

Norepinephrine (NE) stimulated FRTL-5 thyroid cells via an α1-adrenergic receptor, resulting in cytosolic Ca2+ ([Ca2+]i) mobilization and activation of phospholipase C. Adenosine and its receptor agonist, phenylisopropyladenosine (PIA), although not exerting a direct effect, markedly enhanced the NE-induced changes. Basal NE action was not totally abolished whereas the permissive action of adenosine and PIA was completely abolished by pretreatment of the cells with islet-activating protein (IAP), pertussis toxin. The decrease in cAMP level induced by adenosine or PIA is not the cause of their permissive effect, since this effect was not reversed by the addition of cAMP-increasing agents. We conclude that an IAP substrate GTP-binding protein(s) plays a novel role in forming a stimulatory coupling between an adenosine receptor and an α1-adrenergic receptor-coupled phospholipase C system.

Cellular Tolerance to Adenosine Receptor-Mediated Inhibition of Lipolysis: Altered Adenosine 3',5'- Monophosphate Metabolism and Protein Kinase Activation*

Prolonged exposure of many types of cells to drugs or hormones that inhibit the activity of the enzyme adenylate cyclase, such as narcotics and alpha 2-adrenergic agonists, leads to enhanced accumulation of cAMP upon removal of the inhibitory drug. We have found previously that chronic infusion of the adenosine A1 receptor agonist phenylisopropyladenosine (PIA), an inhibitor of adenylate cyclase, into rats leads to enhanced isoproterenol-stimulated cAMP accumulation in adipocytes isolated from these animals. The enhanced cAMP accumulation was associated with an impaired ability of PIA to inhibit lipolysis in these cells. In the present study we have investigated the mechanism of the enhanced cAMP accumulation in adipocytes from PIA-infused rats and the relationship of these changes to the impaired antilipolytic action of the drug. The enhanced isoproterenol-stimulated cAMP accumulation in adipocytes prepared from PIA-infused rats was due to both an increased rate of cAMP synthesis and a decreased rate of cAMP metabolism at high concentrations of cAMP without a change in phosphodiesterase activity. There was heterologous desensitization of the ability of PIA, prostaglandin E1, and nicotinic acid to inhibit cAMP accumulation in the adipocytes from PIA-infused rats; there was an increase in the EC50 of each of these agonists, although maximal inhibition of cAMP accumulation was similar. The relationship between the activation of cAMP-dependent kinase and extent of lipolysis was similar in the two groups of cells. We demonstrated that the explanation for the impaired ability of PIA to decrease the rate of isoproterenol (10(-7) M)-stimulated lipolysis in the cells from the PIA-infused rats was due to the markedly increased concentrations of cAMP in these cells, which led to sufficient activation of the kinase to maintain a high rate of lipolysis even in the presence of PIA. In addition, we found that the changes induced by the PIA infusion were largely reversible over a 2-day period after discontinuing the PIA infusion. These results demonstrate that adipocytes from PIA-infused rats provide an interesting model to investigate the mechanisms of tolerance to inhibitory drugs.

Formula omitted] attenuation of isoproterenol (ISO) enhanced myocardial contractility by phenylisopropyladenosine (PIA) in the rat : F.D. Romano, T.S. Naimi, J.G. Dobson, Jr. Dept. of Physiology, Univ of Massachusetts Med Sch, Worcester, MA. 01655 USA

formula omitted] attenuation of isoproterenol (ISO) enhanced myocardial contractility by phenylisopropyladenosine (PIA) in the rat : F.D. Romano, T.S. Naimi, J.G. Dobson, Jr. Dept. of Physiology, Univ of Massachusetts Med Sch, Worcester, MA. 01655 USA

Adenosine analogs inhibit fighting in isolated male mice

The potent adenosine analogs N-ethylcarboxamide adenosine (NECA) and phenylisopropyladenosine (PIA) inhibit fighting and associated agonistic behaviors in isolated male mice. These effects are reversed by methylxanthines; moderate doses of NECA which inhibit fighting have minimal effects on spontaneous locomotor activity. At very low doses, both NECA and PIA increase fighting in parallel with previously reported increases of motor activity (1–3). Brain levels of [ 3H]-NECA and [ 3H]-PIA achieved at behaviorally effective doses suggest an involvement of adenosine receptors. The biochemical mechanism of adenosine receptor action with respect to fighting is unknown, but may include neuromodulatory effects on the release of other, more classical neurotransmitters.

Dissociation between adenosine receptors and adenylate cyclase in the smooth muscle of guinea pig myometrium

We have previously demonstrated that adenosine causes contraction of guinea-pig myometrium in a fashion consistent with the presence of a purinergic receptor of the A 1 subtype. Incubation of guinea-pig uterine smooth muscle membranes with the stable adenosine analogue [ 3H]cyclohexyladenosine ([ 3H]CHA) resulted in rapid, reversible association of radioligand to saturable sites. The affinity ( K D ) of the receptor for [ 3H]CHA determined from kinetic experiments (3.14 nM) is in good agreement with that determined in saturation experiments ( K D =4.5 nM). Scatchard analysis of specific [ 3H]CHA binding ( B max=79 fmol/mg protein) is consistent with a single class of binding sites for [ 3H]CHA. Computer analysis of competition of [ 3H]CHA binding by the stereoisomers of phenylisopropyl adenosine, R-PIA ( K I =5.3 nM) and S-PIA ( K I =69 nM), as well as the 5′-substituted analogue, ethylcarboxamide adenosine (NECA; K I =4.2 nM) suggest that [ 3H]CHA binding occurs to a single class of receptors of the A 1 subtype. Contractile studies employing these agents reveal that the relative order of potency, based on ED 50 values, correlates well with the relative order of competition of agonist binding, based on equilibrium binding constants. Direct assay of myometrial adenylate cycloase failed to show that adenosine receptors in this smooth muscle are coupled to adenylate cyclase. We conclude here that a smooth muscle adenosine receptor is not coupled to adenylate cyclase, yet subserves muscle contraction. These data are important in light of recent attempts to classify adenosine receptors as dual regulators of adenylate cyclase.

Adenosine receptor-dependent modulation of inhibin secretion in cultured immature rat Sertoli cells

Inhibitory (A1) adenosine receptors that attenuate adenylate cyclase activity are present in cultured Sertoli cells. To investigate the possible effect of activating these receptors on the secretion of inhibin by the Sertoli cell, immature rat Sertoli cells were incubated for 24 h with follicle-stimulating hormone (FSH) in the absence or presence of the non-metabolizable, adenosine agonist phenyl-isopropyl-adenosine (PIA), and the accumulation of α-inhibin immunoreactivity was measured in the medium. Although devoid of effects when added alone, PIA inhibited the FSH-dependent secretion of α-inhibin in a concentration-dependent manner (ED n50 = 1–1.5 nM). PIA treatment of the Sertoli cells also rendered the cells less sensitive to FSH in terms of α-inhibin secretion. The concentration-response curve to FSH was shifted to the right when cells were incubated in the presence of 100–1000 nM PIA. In contrast, dibutyryl cAMP stimulation of α-inhibin accumulation was unaffected by treatment with PIA, indicating that the site of PIA action is at the level of cAMP synthesis. These data provide experimental evidence of adenosine modulation of inhibin secretion by the Sertoli cell and suggest that adenosine may act as a local modulator within the pituitary-testicular axis.

Adenosine inhibition of the hormonal response in the Sertoli cell is reversed by pertussis toxin.

The rat Sertoli cell in culture expresses A1 inhibitory adenosine receptors. In this study, we have used pertussis toxin as a tool to characterize the mechanism of action of adenosine on these cells. Cells were preincubated for 18-24 h with pertussis toxin, and the responses to FSH and to the adenosine analog phenylisopropyladenosine (PIA) were measured by assaying cAMP accumulation. The effect of toxin on adenosine receptors was also evaluated by measuring binding of the adenosine agonist cyclohexyladenosine (CHA). The total number of specific CHA-binding sites was reduced 60-70% in membranes prepared from cells cultured for 24 h in the presence of pertussis toxin; the binding sites remaining after treatment displayed no apparent change in affinity for [3H]CHA. The effect of guanine nucleotides on CHA binding was also reduced after toxin pretreatment, but not abolished. PIA inhibited FSH-stimulated cAMP accumulation by 70-80%. Maximal inhibition was observed at a concentration of 10 nM PIA, and the ED50 of the dose-response curve was 1 nM. Pretreatment of the Sertoli cell with pertussis toxin completely blocked the PIA inhibition. The pertussis toxin effect was time and dose dependent. Reversal of the inhibition was observed after 6 h of treatment with a maximal dose of toxin (100 ng/ml). The dose of toxin producing a half-maximal effect was 10-30 ng/ml. In addition to this blockade of purine nucleotide inhibitory effects, exposure of the Sertoli cell to pertussis toxin concentrations ranging from 1-400 ng/ml consistently led to a potentiation of the FSH response measured as cAMP accumulation. In cell-free preparations (crude particulate fraction of the Sertoli cells, or sucrose gradient-purified plasma membranes), pertussis toxin catalyzed the incorporation of [32P]ADP ribose into a polypeptide with a molecular mass of 40-41 K. This peptide had electrophoretic mobility similar to that of a partially purified guanine nucleotide-binding protein (Gi). These data indicate that adenosine A1 inhibitory receptors are coupled to an inhibitory component (Gi) of adenylate cyclase. In the Sertoli cell, inhibitory and stimulatory signals interact in a bimodal regulation of adenylate cyclase and intracellular cAMP.

Inhibition of the effects of adenosine on force of contraction and the slow calcium inward current by pertussis toxin is associated with myocardial lesions.

The effects of adenosine and the adenosine receptor agonist (-)-N(6)-phenyl-isopropyladenosine (PIA) in the presence of isoprenaline on isometric force of contraction and calcium dependent slow action potentials were studied in papillary muscles from guinea pigs pretreated with pertussis toxin and control guinea pigs. Hearts from guinea pigs treated in the same way with pertussis toxin or solvent alone underwent histological examination. For comparison, hearts from isoprenaline treated guinea pigs were also studied. Pertussis toxin specifically inactivates guanine nucleotide binding proteins (N proteins) involved in transmembrane signal transduction in many receptor systems (for example, adenosine receptors, m-cholinoceptors, and and alpha 2 adrenoceptors). In papillary muscles from control guinea pigs adenosine and PIA in the presence of isoprenaline produced a negative inotropic effect and inhibited the maximal rate of depolarisation of slow calcium dependent action potentials in potassium depolarised papillary muscles. After pretreatment with pertussis toxin the inhibitory effects both on force of contraction and on the maximal rate of depolarisation of adenosine and PIA were abolished. Treatment with pertussis toxin produced disseminated myocardial necrosis and a disseminated cellular calcium overload evidenced by glyoxal-2-bis-hydroxyanil (GBHA) staining. Similar lesions (for example, myocardial necrosis and cellular calcium overload) were also observed after treatment with isoprenaline. In controls neither myocardial necrosis nor cellular calcium overload was found. It is concluded that pertussis toxin sensitive N proteins are involved in the inhibitory effects of adenosine and PIA on force of contraction and on slow calcium inward current during beta adrenergic stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of adenylate cyclase in rat fat cells promotes an increase in GTP content which controls the enzyme activity

In previous studies, we demonstrated that the treatment of adipocytes with cholera toxin or Bordetella pertussis toxin (IAP) promoted an increase in the total guanosine triphosphate (GTP) content of the cells concomitant with the increase in cyclic adenosine monophosphate (AMP) level and the resulting lipolysis. In the present studies, we show that the acute challenge of fat cells with 1 μM isoproterenol (IPNE) is associated with a transient increase in GTP level (3-fold in 6 min). This increase may be attributed to an inhibition of the disposal of GTP or to a stimulation of its synthesis. To evaluate the actual role of GTP, we used virazole, an antitumor agent which inhibits inosinic acid dehydrogenase. After 2 h preincubation of the cells with 1 mM virazole, the effect of a 6 min challenge with 1 μM IPNE is decreased by 59% at the GTP level and by 42% in cyclic AMP production. One hour later, the resulting lipolytic efficiency is reduced by 57%. IAP treatment (10 μg/ml) produced its maximal effect on GTP and cyclic AMP levels and on lipolysis after 90 min incubation. The antilipolytic effect of 1 μM phenylisopropyladenosine (PIA) is almost abolished. When 1 mM virazole is added to the cell suspension to deplete the guanyl nucleotide pool, the resulting lipolysis due to IAP treatment is decreased by 85%, whereas GTP and cyclic AMP levels were decreased by 80 and 70%, respectively. We can conclude that the cyclic AMP synthesis in intact cells is accompanied by a parallel increase of their GTP content, whether the stimulation results from the activation of G s or the inhibition of G i. The reduction of the guanyl nucleotide pool under virazole results in a relatively less important inhibition of lipolysis when G s is stimulated than when it is G i.

Lowering of plasma glucose in diabetic rats by antilipolytic agents.

Both nicotinic acid (NA) and the adenosine receptor agonist phenylisopropyladenosine (PIA) are potent antilipolytic agents. We have evaluated the ability of these compounds to lower plasma glucose concentration in 450-g male diabetic rats. Diabetes was induced by intravenous streptozotocin, and the rats were studied 7-10 days later. Mean (+/- SE) fasting glucose decreased 4 h after subcutaneous injections of PIA at 0 and 2 h. A similar change in plasma glucose level was also seen in rats injected with NA. The decrease in the concentration of plasma glucose in both instances was preceded by marked sustained reductions in plasma free fatty acid (FFA) concentrations; FFA decreased in PIA-injected rats and in response to NA. With injection of normal saline, neither plasma glucose nor FFA concentrations decreased in diabetic rats. There was no change in the plasma insulin concentration of rats that had hypoglycemic responses to PIA or NA. In vitro glucose uptake was determined in isolated adipocytes, and both PIA and NA were shown to increase basal and maximal insulin-stimulated glucose uptake. The stimulating effect of the two compounds was similar, and the magnitude of the effect was comparable in adipocytes from either normal or diabetic rats. As a result, neither NA nor PIA could restore the defects in glucose transport to normal in adipocytes from diabetic rats. Insulin-stimulated glucose uptake was assessed in vivo by determining the steady-state glucose response of diabetic rats to a continuous infusion of insulin and glucose and was found to be significantly enhanced in response to NA compared with NaCl.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of glycogenolysis in isolated hepatocytes by adenosine and one of its analogues is inhibited by caffeine.

The adenosine analogues 5'-(N-ethyl)carboxamidoadenosine (NECA) and N6-(phenylisopropyl)adenosine (PIA) activate glycogen phosphorylase 5-fold and 4.2-fold respectively in rat hepatocytes incubated in the absence of endogenous adenosine. Half-maximally effective concentrations are 0.5 microM for NECA and 20 microM for PIA, demonstrating the presence of A2-adenosine receptors. Exogenous adenosine activates phosphorylase 4.6-fold, but high rates of adenosine disappearance from the medium render estimates of its half-maximally effective concentration unreliable. These effects of NECA and adenosine are inhibited by 0.1 mM-caffeine. Activation of phosphorylase by a physiological concentration of adenosine (3.3 microM) was 50% inhibited by a physiological concentration of caffeine (35 microM).

Presence of a functional inhibitory GTP-binding regulatory component, Gi, linked to adenylate cyclase in adipocytes of ob/ob mice.

It has been reported recently (Begin-Heick, N. (1985) J. Biol. Chem. 260, 6187-6193) that adipocytes from the obese mouse strain (ob/ob), unlike normal mice (+/+), lack functional Gi, a GTP-regulated protein complex that mediates inhibition of adenylate cyclase. In contrast, we have found functional Gi linked to inhibition of adenylate cyclase in adipocyte membranes from both ob/ob and +/+ mice. This conclusion is based on observation of: 1) GTP-dependent inhibition of adenylate cyclase by antilipolytic agents, such as prostaglandin E2, nicotinic acid, and the adenosine receptor agonist, phenylisopropyladenosine (PIA); 2) classical biphasic GTP kinetics, with stimulation by low and inhibition by high concentrations of GTP; and 3) elimination of cyclase inhibition by antilipolytic agents upon treatment of ob/ob adipocytes with pertussis toxin. Upon treatment with pertussis toxin and [32P] NAD, purified adipocyte membranes from ob/ob mice incorporated twice as much radioactivity per unit membrane protein than those from +/+ mice in the 40,000-42,000 region. The inhibitory actions of PIA on adenylate cyclase were blocked by the adenosine receptor antagonists, theophylline and isobutylmethylxanthine. However, in contrast to other known inhibitory adenosine receptors, relatively high (100 nM) PIA concentrations were required for half-maximal inhibition of adenylate cyclases from both +/+ and ob/ob adipocytes. The adipocyte adenylate cyclase from both mouse strains were approximately equally susceptible to inhibition by nicotinic acid and prostaglandin E2. However, the ob/ob cyclase was inhibited by 47% with PIA, whereas the enzyme from the +/+ mouse was inhibited by only 27% (p less than 0.01). This greater inhibition by adenosine may contribute to abnormal fat metabolism in adipocytes from ob/ob mice.